Construction of co-integrate plasmids from plasmids of Streptomyces and Escherichia

ABSTRACT

Novel chemical compounds, recombinant plasmids pUC1019 and pUC-1020, which are obtained by covalent linkage of ca. 4.2 kb BclI restriction endonuclease fragment of the Streptomyces espinosus plasmid pUC6 into the BamHI endonuclease site of the E. coli plasmid pBR322. Plasmid pUC1024 is obtained by restructuring plasmid pUC1019. These plasmids are useful as cloning vehicles in recombinant DNA work. For example, using DNA methodology, a desired gene, for example, the insulin gene, can be inserted into the plasmids and the resulting plasmids can then be transformed into a suitable host microbe which, upon culturing, produces the desired insulin.

BACKGROUND OF THE INVENTION

The development of plasmid vectors useful for recombinant DNA geneticsamong microoganisms is well known. The editorial in Science, Vol. 196,April, 1977, gives a good summary of DNA research. This editorial isaccompanied by a number of supporting papers in the same issue ofScience.

Similar DNA work is currently being done on industrially importantmicroorganisms of the genus Streptomyces. [Bibb, M. J., Ward, J. M., andHopwood, D. A. 1978. "Transformation of plasmid DNA into Streptomyces athigh frequency." Nature 274, 398-400.] Though plasmid DNA's have beendetected in several streptomycetes [Huber, M. L. B. and Godfrey, O.1978. "A general method for lysis of Streptomyces species." Can. J.Microbiol. 24, 631-632.] [Schrempf, H., Bujard, H., Hopwood, D. A. andGoebel, W. 1975. "Isolation of covalently closed circulardeoxyribonucleic acid from Streptomyces coelicolor A3(2)." J. Bacteriol.121, 416-421.] [Umezawa, H. 1977. "Microbial secondary metabolites withpotential use in cancer treatment (Plasmid involvement in biosynthesisand compounds)." Biomedicine 26, 236-249.], [Malik, V. S. 1977.Preparative Method for the isolation of super-coiled DNA from achloramphenicol producing streptomycete. J. Antibiotics 30, 897899],only a few streptomycete plasmids have been physically isolated andextensively characterized [Schrempf, supra]. See also [Bibb, M.,Schottel, J. L., and Cohen, S. N. 1980. A DNA cloning system forinterspecies gene transfer in antibiotic-producing Streptomyces. Nature284, 526-531.] and [Thompson, C. J., Ward, J. M. and Hopwood, D. A.1980. DNA cloning in Streptomyces:resistance genes fromantibiotic-producing species. Nature 286, 525-529.] The existence ofother plasmids in the genus Streptomyces has been inferred from reportedgenetic data as follows:

(1) Akagawa, H., Okanishi, M. and Umezawa, H. 1975. "A plasmid involvedin chloramphenicol production in Streptomyces venezuelae: Evidence fromgenetic mapping." J. Gen. Microbiol. 90, 336-346.

(2) Freeman, R. F. and Hopwood, D. A. 1978. "Unstable naturallyoccurring resistance to antibiotics in Streptomyces." J. Gen. Microbiol.106, 377-381.

(3) Friend, E. J., Warren, M. and Hopwood, D. A. 1978. "Genetic evidencefor a plasmid controlling fertility in an industrial strain ofStreptomyces rimosus." J. Gen. Microbiol. 106, 201-206.

(4) Hopwood, D. A. and Wright, H. M. 1973. "A plasmid of Streptomycescoelicolor carrying a chromosomal locus and its inter-specifictransfer." J. Gen. Microbiol. 79, 331-342.

(5) Hotta, K., Okami, Y. and Umezawa, H. 1977. "Elimination of theability of a kanamycin-producing strain to biosynthesizedeoxystreptamine moiety by acriflavine." J. Antibiotics 30, 1146-1149.

(6) Kirby, R., Wright, L. F. and Hopwood, D. A. 1975."Plasmid-determined antibiotic synthesis and resistance in Streptomycescoelicolor." Nature 254, 265-267.

(7) Kirby, R. and Hopwood, D. A. 1977. "Genetic determination ofmethylenomycin synthesis by the SCPI plasmid of Streptomyces coelicolorA3(2)." J. Gen. Microbiol. 98, 239-252.

(8) Okanishi, M., Ohta, T. and Umezawa, H. 1969. "Possible control offormation of aerial mycelium and antibiotic production in Streptomycesby episomic factors." J. Antibiotics 33, 45-47.

Plasmid pUC6 was isolated from Streptomyces espinosus biotype 23724a,NRRL 11439.

Plasmid pBR322 is a well known plasmid which can be obtained from E.coli RR1, NRRL B-12014. The restriction endonuclease map for pBR322 ispublished; Sutcliff, J. G. "pBR322 restricting map derived from the DNAsequence: accurate DNA size markers up to 4361 nucleotide pairs long."Nucleic Acids Research 5, 2721-2728, 1978. This map is incorporatedherein by reference to the above publication.

BRIEF SUMMARY OF THE INVENTION

Plasmids pUC1019 and pUC1020 are obtained by the in vitro linkage of˜4.2 kb BclI restriction endonuclease fragment of the S. espinosusplasmid pUC6 into the BamH1 endonuclease site of the E. coli plasmidpBR322.

Plasmids pUC1019 and pUC1020 constitute the insertion of this BclIfragment in the two possible orientations in pBR322. In a like manner,the 4.1 and 0.9 kb BclI restriction fragments of pUC6 can be recombinedwith pBR322.

The invention further includes the construction of plasmid pUC-1024,which is derived from pUC1019 by in vitro deletion of DNA sequencesbetween endonuclease PvuII sites. Plasmid pUC1024 is ˜3.5 kilobases (kb)smaller than pUC1019.

The plasmids, advantageously, are transformed into a suitable host, forexample, E. coli.

Plasmids pUC1019 and pUC1020 are recombinant DNA molecules consisting ofthe entire genome of the small (˜2.6×10⁶ dalton) high copy number(˜30/chromosome) E. coli plasmid pBR322 and ˜46% (4.2 kb) of the genomeof the small (˜6.0×10⁶ daltons) high copy number (˜30/chromosome) S.espinosus plasmid pUC6. Plasmids pUC1019 and pUC1020 contain singlesites for the restriction endonucleases EcoRI, PstI, HindIII and XhoI.The XhoI site will also allow the cloning of SalI restriction fragments.Hence plasmids pUC1019 and pUC1020 represent DNA molecules which mayfunction as vectors in both E. coli and Streptomyces and representvaluable intermediates for the development of better host-vectorsystems.

Plasmids pUC1019, pUC1020, and pUC1024 are characterized by therestriction maps shown in FIGS. 2, 3, and 5, respectively, of thedrawings. The restriction endonuclease abbreviations shown in thedrawings are standard and well known in the art.

Plasmid pUC6 is obtainable from the novel microorganism Streptomycesespinosus biotype 23724a, NRRL 11439. This plasmid can be obtained fromNRRL 11439 by growing the culture on a suitable medium, fragmenting themycelia, incubating the fragmented mycelia, harvesting the culture aftera suitable time, and then lysing the mycelia. From this lysate it ispossible to isolate essentially pure pUC6. pUC6 is characterized bystandard characterization tests which include its molecular weight,approximately 6.0 megadaltons, sensitivity to restriction endonucleases,infra, and presence at 20-40 copies per S. espinosus NRRL 11439 cell.

REFERENCE TO THE DRAWINGS

FIG. 1--This shows the isolation scheme of the plasmids.

FIG. 2--Restriction endonuclease cleavage map for pUC1019.

FIG. 3--Restriction endonuclease cleavage mpa for pUC1020.

FIG. 4--Construction of recombinant plasmid pUC1024.

FIG. 5--Restriction endonuclease cleavage map for pUC1024.

The maps are constructed on the basis of plasmids pUC1019 and pUC1020having a molecular weight of ca. 5.7 megadaltons or a molecular lengthof ca. 8.6 kilobases (kb). Plasmid pUC1024 has a molecular length of ca.5.1 kb. It retains the locus conferring genetic instability to plasmidpUC1019 in E. coli hosts. Plasmid pUC1024 has single restriction sitesfor the endonucleases EcoRI, HindIII, PstI and PvuII. The PvuII sitewill allow the cloning of blunt ended DNA fragments from a wide range ofrestriction enzyme digests or from other properly prepared DNAfragments. The restriction endonuclease abbreviations are as follows:(1) BglII is an enzyme from Bacillus globigii; (2) BclI is an enzymefrom Bacillus caldolyticus; (3) PvuII is an enzyme from Proteusvulgaris; and (4) XhoI is an enzyme from Xanthomonas holicola.

pUC1019, pUC1020, and pUC1024, can be used to create recombinantplasmids which can be introduced into host microbes by transformation.The process of creating recombinant plasmids is well known in the art.Such a process comprises cleaving the isolated vector plasmid at aspecific site(s) by means of a restriction endonuclease, for example,BglII, XhoI, and the like. The plasmid, which is a circular DNAmolecule, is thus converted into a linear DNA molecule by the enzymewhich cuts the two DNA strands at a specific site. Other non-vector DNAis similarly cleaved with the same enzyme. Upon mixing the linear vectoror portions thereof and non-vector DNA's, their single-stranded or bluntends can pair with each other and in the presence of a second enzymeknown as polynucleotide ligase can be covalently joined to form a singlecircle of DNA.

The above procedure also can be used to insert a length of DNA from ahigher animal into pUC1019, pUC1020, or pUC1024. For example, the DNAwhich codes for ribosomal RNA in the frog can be recombined with pUC1019DNA. The resulting circular DNA molecules consist of plasmid pUC1019with an inserted length of frog rDNA.

The recombinant plasmids containing a desired genetic element, preparedby using pUC1019, pUC1020, or pUC1024, can be introduced into a hostorganism for expression. Examples of valuable genes which can beinserted into host organisms by the above described process are genescoding for somatostatin, rat proinsulin, interferon, and proteases.

The usefulness of plasmids pUC1019, pUC1020, and pUC1024 is derived fromtheir capacity to function as plasmid vectors in industrially importantmicroorganisms, e.g., Streptomyces. Also, pUC1019, pUC1020, and pUC1024are especially useful because of their single restriction sites. Hence,cloning of genetic information from Streptomyces into pUC1019, pUC1020,or pUC1024 provides a means of increasing the production of commerciallyimportant products from these organisms, e.g., antibiotics.

This approach is compared to the concept of cloning genes for antibioticproduction into the well characterized Escherichia coli K-12 host-vectorsystem. The E. coli system has the disadvantage that it has been foundthat genes from some Gram-positive organisms, e.g., Bacillus, do notexpress well in the Gram-negative E. coli host. Likewise, plasmids fromGram-negative organisms are not maintained in Gram-positive hosts, andGram-negative genetic information is either expressed poorly or not atall in Gram-positive hosts. This clearly argues for the advantage of aGram-positive host-vector system and argues the usefulness of plasmidpUC1019, pUC1020, or pUC1024, in such a system.

In general, the use of a host-vector system to produce a product foreignto that host requires the introduction of the genes for the entirebiosynthetic pathway of the product to the new host. As discussed above,this may lead to problems of genetic expression, but may also generatenew and/or increased problems in the fermentation of the microorganismsand in the extraction and purification of the product. A perhaps moreuseful approach is to introduce a plasmid vector into a host whichnormally produces the product and clone onto that plasmid the genes forbiosynthesis of the product. At the very least, problems of fermentationand product extraction and purification should be minimized.Additionally, in this cloning system it may not be necessary to cloneand amplify all the genes of the biosynthetic pathway, but rather it maybe necessary only to clone regulatory genes or genes coding for theenzymes that are rate limiting in product biosynthesis. Since pUC1019and pUC1020 are cointegrate plasmids, they can be used to clone DNAsequences in E. coli or within the genera of Streptomyces andMicromonospora, as well as within other microbes.

DETAILED DESCRIPTION OF THE INVENTION The Microorganisms and Plasmids

The following microorganisms are available from the permanent collectionof the Northern Regional Research Laboratory, U.S. Department ofAgriculture, Peoria, Ill., U.S.A.

NRRL B-12110--E. coli CSH50

NRRL B-11439--S. espinosus biotype 23724a

NRRL B-12014--E. coli RR1 (pBR322)

NRRL B-12252--E. coli CSH50 (pUC1019)

NRRL B-12253--E. coli CSH50 (pUC1020)

NRRL B-12254--E. coli RR1 (pUC1024)

NRRL B-12186--E. coli RR1

These deposits are available to the public upon the grant of a patent tothe assignee, The Upjohn Company, disclosing them. The deposits are alsoavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny, are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by governmental action.

The following examples are illustrative of the process and products ofthe subject invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Isolation of Vector pBR322 DNA from E. coli NRRL B-12014

A 100 ml. culture of E. coli RR1 (pBR322) is grown overnight in L-brothwhich consists of the following ingredients:

    ______________________________________                                        Bacto tryptone (Difco)                                                                           10 g./liter                                                Bacto yeast extract (Difco)                                                                      5 g./liter                                                 NaCl               5 g./liter                                                 Ampicillin         50 mg./liter                                               ______________________________________                                    

The cells are recovered by centrifugation at 17,000×g. for 10 minutes ina refrigerated centrifuge. The pellet is suspended in 2.5 ml. 50 mM trisbuffer (pH 8) containing 25% sucrose. One-half ml. of lysozyme stocksolution is added (5 mg./ml. in TES buffer). The mixture is allowed tostand in ice for 5 minutes. At this point 1 ml. EDTA (0.25 M, pH 8) isadded and the mixture is again allowed to stand in ice for 5 minutes.One and a quarter ml. of 5 N NaCl and 1 ml. 10% SDS (sodium dodecylsulfate) are then added. The mixture is shaken on a Vortex and incubatedat 37° C. for 20 minutes. Then 10 μl of ribonuclease (20 mg./ml.) isadded and the sample is again incubated at 37° C. for 20 minutes. Themixture is then kept in ice overnight and then centrifuged at 35,000×g.for 30 minutes in a refrigerated centrifuge. 2 ml. of the supernatantsolution (lysate) are carefully removed with a pipette. Four andone-half ml. of TES buffer (30 mM tris.HCl, pH 8, 5 mM EDTA.Na₂, 50 mMNaCl) are mixed with 1.5 ml. EtBr (ethidium bromide) stock (1 mg./ml. inTES buffer) and 7.5 g. solid CsCl. After the salt has dissolved, 2 ml.of the lysate, described above, is added and the mixture is transferredinto a polyallomer tube fitting a titanium 50 (50 Ti) head (Beckmanultracentrifuge). The tubes are filled to the top with mineral oil andcentrifuged in a Beckman ultracentrifuge at 40,000 rpm in a 50 Ti headat 15° C. for at least 2 days. The DNA is located under a long waveUV-lamp and the heavier band containing the plasmid DNA is removed witha syringe by puncturing the tube wall from the side. The samples areextensively dialysed against 200 volumes of TES buffer at 4° C.Following dialysis, 1/10 sample volume of a 3 M Na acetate stocksolution is added and the plasmid DNA is precipitated by the addition of2 volumes of cold ethanol. The resulting pellet is then lyophilized andredissolved in 200 μl 10 mM tris buffer, pH 7.8 containing 1 mM EDTA.Na₂and frozen for storage.

EXAMPLE 2 Isolation of Plasmid pUC6 from a Biologically Pure Culture ofStreptomyces espinosus, biotype 23724a, NRRL 11439

The spores from a biologically pure culture of Streptomyces espinosusbiotype 23724a, NRRL 11,439, are inoculated into 10 ml. of the followingDifco Antibiotic Medium No. 3 Broth (Difco Labs., Detroit, Mich.): 0.15%Beef extract; 0.15% yeast extract; 0.5% peptone; 0.1% glucose; 0.35%NaCl; 0.368% K₂ HPO₄ ; 0.132% KH₂ PO₄.

The medium has previously been sterilized in a 50 ml. Erlenmeyer flask.After inoculation, the flask is incubated at 37° C. for about 36 to 48hours on a Gump or New Brunswick rotary shaker operating at 100-250 rpm.Upon completion of the incubation, the mycelia-broth suspension in theflasks is homogenized under sterile conditions and is then mixed in asterile 125 ml. Erlenmeyer flask containing 10 ml. of the above mediumand also, advantageously 68% (w/v) sucrose and 1% (w/v) glycine. Theaddition of sucrose and glycine facilitates the subsequent lysing of thecells. The amounts of sucrose and glycine in the medium can be varied byroutine adjustments with the goal being to facilitate the subsequentlysing of the cells. The flask is then incubated further for another 36to 48 hours at 37° C. on a Gump rotary shaker, as above. After thisincubation, the mycelia are separated from the broth by low speedcentrifugation, for example, at 6000×g. for 15 minutes at 4° C. anddecantation of the supernatant from the mycelial pellet.

The supernatant is discarded and the pellet is resuspended in 1.5 ml. ofan isotonic buffer containing ethylenediaminotetraacetic acid (EDTA) andsucrose, e.g., TES buffer [0.03 M tris(hydroxymethyl)aminomethane(Tris), 0.005 M EDTA and 0.05 M NaCl; pH=8.0] containing 20% (w/v)sucrose. Next, 1.5 ml. of a 5 mg./ml. solution of lysozyme in the samebuffer is added and the mixture is incubated at 37° C. for 30 minuteswith occasional mixing. Then, 1.5 ml. of 0.25 M EDTA (pH=8.0) is addedand this mixture is incubated 15 minutes at 37° C. Subsequently, thecell suspension is lysed by the addition of 2.5 ml. of a lytic mixture,e.g. [1.0% (w/v) Brij-58 (a detergent sold by Pierce Chem. Co.,Rockford, Ill.), 0.4% (w/v) deoxycholic acid, 0.05 M Tris (pH=8.0) and0.06 M EDTA] and incubation of this mixture at 37° C. for 20 minutes.The lysate is then sheared by passing it 5-10 times through a 10 ml.pipette. The sheared lysate is then digested with ribonuclease (140μg/ml.) and pronase (300 μg/ml.) for an additional 20 minutes at 37° C.Alternatively, the cell-lysozyme-EDTA mixture can be digested withribonuclease and pronase before lysis with a lytic agent such as 2%sodium dodecyl sulfate in water.

This crude lysate material is then mixed with a salt, for example,cesium chloride (preferred), and cesium sulfate, and the intercalatingdye ethidium bromide to give a solution of density ρ=1.550. Thissolution is centrifuged to equilibrium at 145,000×g. (isopycnic densitygradient centrifugation). The covalently closed circular plasmid DNA isthen visible in the centrifuge tube under long wave ultraviolet (320 nm)illumination as a faint fluorescent band below the intensely fluoresentband of linear chromosomal and plasmid DNA's.

Covalently closed circular plasmid DNA is prepared for characterizationby removing it from the isopycnic gradients, extracting the ethidiumbromide by two treatments with one-third volume of isopropyl alcohol andthen dialyzing the aqueous phase against an appropriate buffer, e.g.,0.1×SSC buffer (0.015 M NaCl, 0.0015 M sodium citrate; pH=7.4) to yieldessentially pure pUC6.

Characteristics of pUC6

Molecular Weight: ca. 6.0 megadaltons

Copies per Cell: 20-40

Restriction Endonuclease Sensitivities: pUC6 has the followingsensitivies to restriction endonucleases.

    ______________________________________                                        Plasmid Sensitivities to Restriction Endonucleases                            # Cleavage Sites   # Cleavage Sites                                           Enzyme    pUC6         Enzyme   pUC6                                          ______________________________________                                        BglI      >7           BglII    1                                             BamHI     0            HpaI     0                                             HpaII     Many         HindIII  0                                             EcoRI     0            KpnI     0                                             PstI      0            PvuII    4                                             MboII     >5           AvaI     >7                                            XbaI      0            XhoI     2                                             SalI      5-6          SmaI     >5                                            HincII    >7           BclI     3                                             ______________________________________                                    

These results were obtained by digestion of pUC6 DNA in the presence ofan excess of restriction endonuclease. The number of restriction siteswere determined from the number of resolvable fragments in either 0.7 or1.0% agarose gels.

EXAMPLE 3 Preparation of Plasmids pUC1019 and pUC1020

Plasmids pUC6 and pBR322, prepared as described above, are linearized bydigestion with restriction endonucleases BclI and BamHI respectively.Plasmid pBR322 DNA is digested with BamHI restriction enzyme by mixing˜50 μl. of DNA (˜0.5 μg) solution in TE buffer (0.01 M Tris .HCl, 0.001M Na₂ EDTA; pH 8.0) with 50 μl of 2X restriction buffer (Post et al.,Cell 15, 215-229, 1978) and 4 units of BamHI enzyme preparation. Thismixture is incubated at 37° C. for 1 hour. The digest is then applied toa 1% preparative low melting point agarose gel and electrophoresed for˜3 hours at 50 volts and 4° C. The resolved DNA fragments are visualizedby ethidium bromide staining and long wave ultraviolet lightillumination. The region of the gel containing the DNA is excised fromgel and heated to 65° C. in the presence of 1.5 ml. of TE buffer to meltthe gel and release the DNA from the gel matrix. This suspension ischilled and centrifuged at 37,000×g to pellet the agarose. Thesupernatant is decanted and saved. The agarose pellet is extracted asecond time by heating to 65° C. with TE buffer. The two supernatantsare pooled and ethanol precipitated by the addition of 0.1 volume of NaAcetate and 2 volumes 95% ethanol at -20° C. The DNA precipitate iscollected by centrifugation at 85,000×g at 4° C. for 60 minutes. Theprecipitate is redissolved in 100 μl of TE buffer. This sample is usedfor ligation as described below.

Plasmid pUC6 is subjected to BclI digestion in a reaction mixturecontaining 50 μl of pUC6 DNA (˜0.5 μg) solution in TE buffer and 50 μlof 2X BclI restriction buffer and 4 units of BclI enzyme. This mixtureis incubated at 37° C. for one hour and the digest placed in andisolated from a preparative agarose gel electrophoresis system asdescribed above.

For ligation, 25 μl of BamHI digested pBR322 DNA, 25 μl of BclI digestedpUC6 DNA and 20 μl DD H₂ O are combined. Ten μl 100 mM DDT, 10 μl 50 mMMgCl₂ and 10 μl of 0.5 mM ATP are combined with the restricted DNAmixture. Finally, 1.0 unit of T₄ DNA ligase is added and the sample iskept in ice for 1-2 days.

For transformation into E. coli CSH50, inoculum is grown overnight inL-broth and diluted 1:100 into fresh L-broth the next day. The cells areincubated at 37° C. and allowed to grow to an OD₆₅₀ of 0.2. At thispoint 50 ml. of culture is centrifuged in the cold, the pelletresuspended in 20 ml. cold 100 mM CaCl₂, incubated at 0° C. for 20-25minutes and centrifuged again. The pellet is then resuspended in 0.5 ml.cold 100 mM CaCl₂ solution and kept at 0°-4° C. for 24 hours. (Dagert,M. and Ehrlich, S. D. 1979, Gene 6: 23-28) One hundred μl of pUC1019 andpUC1020 ligase mixture (see above) is mixed with 500 μl cell suspension.This mixture is kept in ice for 10 minutes, and then at 37° C. for 5minutes. Ten to 20 ml. of L-broth is added and the cell suspension isincubated 1-2 hours at 37° C. Next, 100 μl aliquots are plated onfreshly prepared agar plates containing 25 ml. of L-broth, 1.5% agar,and 50 μg of ampicillin/ml. Colonies are selected and scored fortetracycline sensitivity.

Suspected recombinant DNA containing transformants are then grown in 25ml. cultures. Cleared lysates are prepared by pelleting the cells fromthe culture medium at ˜10,000×g. The pellet is resuspended in 10 ml. ofcold TES buffer (30 mM Tirs.HCl, 5 mM Na₂ EDTA and 50 mM NaCl, pH 8.0)and pelleted again. This pellet is resuspended in 1 ml. of TES buffercontaining 20% sucrose. 0.2 ml. of lysozyme solution (5 mg./ml. in TES)is added and incubated on ice 15 minutes at which time 0.4 ml. of 0.25 MNa₂ EDTA (pH 8.0) is added and the incubation continued 15 minutes. 1.6ml. of a lytic mix (1% Brij 58, 0.4% Na deoxycholate, 0.05 M Tris.HCl,62.5 mM Na₂ EDTA; pH 8.0) is added and the lysate incubated anadditional 15 minutes at 4° C. The lysate is sheared by passage 5 timesthrough a 10 ml. pipette. The bulk of the cellular DNA and debris areremoved by centrifugation at 48,000×g for 30 minutes. The cleared lysateis digested successively for 15-minute intervals with pancreatic RNAse A(100 mcg./ml.) and Pronase (200 mcg./ml.) at 37° C. These lysate arethen centrifuged in a CsCl-ethidium bromide isopycnic density gradient.Plasmid DNA isolated from these gradients is characterized by digestionwith restriction endonuclease.

EXAMPLE 4 Preparation of Plasmid pUC1024

Plasmid pUC1019, prepared as described in Example 3, is linearized bydigestion with restriction endonuclease PvuII as follows:

Approximately 0.5 μg of pUC1019 DNA in 25 μl of TE buffer is mixed withan equal volume of 2X PvuII restriction enzyme buffer (0.3 M NaCl, 12 mMTris.HCl [pH 7.4], 12 mM MgCl₂, 12 mM 2-mercaptoethanol) and two unitsof PvuII restriction enzyme. This sample is digested for one hour at 37°C.

The resulting digest is then applied to a 1% preparative low meltingpoint agarose gel and electrophoresed for ˜3 hours at 50 volts and 4° C.The remainder of the agarose gel procedure is as described above inExample 3. The appropriate PvuII fragment is isolated and ligated, asdescribed above in Example 3.

Transformation of the ligated plasmid, which is now plasmid pUC1024,into E. coli RR1 is as described in Example 3.

Recombinant plasmids were characterized by cleavage with restrictionendonucleases.

Restriction endonucleases were obtained as commercial preparations fromMiles Laboratoris, Bethesda Research Laboratories, and New EnglandBiolabs. Enzyme diges-tions were prepared in accordance with theconditions specified by the suppliers using at least a two-fold excessof endonuclease.

The digested samples were applied to 0.7-1% agarose gels and wereelectrophoresed for 2 hours at a constant applied voltage of 10-15 v/cmof gel height. [Sharp, P. A., Sugden, J. and Sambrook, J. 1973.Detection of two restriction endonuclease activities in Haemophilusparainfluenzae using analytical agarose-ethidium bromideelectrophoresis. Biochemistry 12, 3055-3063]. The molecular weights ofrestriction fragments were determined relative to the standard migrationpatterns of bacteriophage lambda DNA digested with enzyme HindIII[Murray, K. and Murray, N. E. 1975. "Phage lambda receptor chromosomesfor DNA fragments made with restriction endonuclease III of Haemophilusinfluenzae and restriction enconuclease I of Escherichia coli." J. Mol.Biol. 98, 551-564] or EcoRI [Helling, R. B., Goodman, H. M. and Boyer,H. W. 1974. Analysis of endonuclease R.EcoRI fragments of DNA fromlambdoid bacteriophages and other viruses by agarose-gelelectrophoresis. J. Virology 14, 1235-1244].

Cointegrate plasmids pUC1019 and pUC1020, and restructured plasmidpUC1024, can be isolated from their E. coli hosts by well knownprocedures, e.g., using the cleared lysate-isopycnic density gradientprocedures described above. Once transformants containing pUC1019,pUC1020, or pUC1024 are identified, they are separated as pure entitiesin a pure culture. These plasmids can be differentiated as distinctentities by their unique restriction patterns as would be predicted bytheir restriction maps.

As shown herein, plasmid pUC1019 can be used as a substrate for in vitrodeletion restructuring, e.g., pUC1019 can be digested with PvuII to forma derivative lacking some of the PvuII restriction sites. Thisadvantageously also gives a smaller plasmid.

Examples of other vectors which can be used in the invention as asubstitute for pBR322 are pBR313, which codes for ampicillin andtetracycline resistance, pSC101, which codes for tetracyclineresistance, pCR11, which codes for kanamycin resistance, λ bacteriophagevectors, for example, charon phages, and yeast 2μ plasmid DNA.

Examples of other hosts for the vectors are any E. coli K-12 derivative[Bacteriological Reviews, December 1972, pages 525-557] (these have beenapproved by the NIH Guidelines) and yeasts, other fungi, or otherbacteria. It is recognized that these latter hosts would also have to beapproved by the NIH Guidelines.

The work described herein was all done in conformity with physical andbiological containment requirements specified in the NIH Guidelines.

We claim:
 1. E. coli CSH50 (pUC1019) having the deposit accession numberNRRL B-12252.
 2. E. coli CSH50 (pUC1020) having the deposit accessionnumber NRRL B-12253.
 3. Cointegrate plasmid pUC1019, characterized asshown by the restriction map in FIG. 2 of the drawings.
 4. Cointegrateplasmid pUC1020 characterized as shown by the restriction map in FIG. 3of the drawings.
 5. A process for preparing cointegrate plasmids pUC1019and pUC1020 which comprises:(a) linearizing plasmid pBR322 by BamHIendonuclease to obtain linear plasmid DNA; (b) linearizing plasmid pUC6by BclI endonuclease to obtain linear plasmid DNA; and (c) ligating saidlinear plasmid DNA from pBR322 and pUC6 to obtain cointegrate plasmidspUC1019 and pUC1020.
 6. A process for cloning ca. 4.2 kb BclIrestriction endonuclease fragment of plasmid pUC6 into a suitable hostwhich comprises:(a) linearizing plasmid pBR322 by BamHI enconuclease toobtain linear plasmid DNA; (b) linearizing plasmid pUC6 by BclIendonuclease to obtain linear plasmid DNA; (c) ligating said linearplasmid DNA from pBR322 and pUC6 to obtain cointegrate plasmids pUC1019and pUC1020; and, (d) transforming said cointegrate plasmids into saidsuitable host.
 7. A process, according to claim 6, wherein said suitablehost is a bacterium.
 8. A process, according to claim 7, wherein saidbacterium is E. coli CSH50.
 9. E. coli RR1 (pUC1024) having the depositaccession number NRRL B-12254.
 10. Plasmid pUC1024, characterized asshown by the restriction map in FIG. 5 of the drawings.
 11. A processfor preparing plasmid pUC1024 which comprises:(a) digestion of pUC1019DNA with restriction endonuclease PvuII to obtain fragmented linearplasmid DNA; and, (b) ligating the PvuII fragment of said plasma DNA toobtain plasmid pUC1024.
 12. A process for cloning plasmid pUC1024 into asuitable host which comprises:(a) digestion of pUC1019 DNA withrestriction endonuclease PvuII to obtain fragmented linear plasmid DNA;(b) ligating the PvuII fragment of said plasmid DNA to obtain plasmidpUC1024; and, (c) transforming said plasmid into said suitable host. 13.A process, according to claim 12, wherein said suitable host is abacterium.
 14. A process, according to claim 13, wherein said bacteriumis E. coli RR1.